Diagnostic and contrast agent

ABSTRACT

The present invention relates to a method of contrast enhanced ultrasonic diagnostic imaging comprising administering to a subject a contrast enhancing amount of spherical particles comprising a matrix enclosing a gaseous contrast agent which reflects sound waves, said matrix being a biocompatible, biodegradable, non-immunogenic non-polyamino acid synthetic polymer; and generating an ultrasonic image of said subject. The polymer may be selected from the group consisting of carbohydrates, carbohydrate derivatives and non-polyamino acid synthetic polymers.

This application is a Continuation of application Ser. No. 08/361.466.filed Dec. 22. 1994. now U.S. Pat. No. 5,618,514 which is a continuationof abandoned application Ser. No. 08/186,096 filed Jan. 25, 1994, whichis a continuation of abandoned application Ser. No. 08/043,982 filedApr. 7, 1993, which is a continuation of abandoned application Ser. No.07/888,305 filed May 27, 1992, which is a continuation of abandonedapplication Ser. No. 07/693,031 filed Apr. 30, 1991, which is acontinuation of abandoned application Ser. No. 07/278,326 filed Nov. 30,1988, which is a continuation of abandoned application Ser. No.06/775,047 filed Aug. 15, 1985.

BACKGROUND

The invention relates to response particles, preferably spheres, andtheir use as a diagnostic and contrast agent.

In diagnostic medicine, contrast agents are today being used primarilyin X-ray diagnostics where an increased contrast effect is obtainedduring examination of, for example, internal organs, such as thekidneys, the urinary tract, the digestive tract, the vascular system ofthe heart (angiography), etc. This contrast effect is based upon thefact that the contrast agent itself is less permeable to X-rays than thesurrounding tissue, as a result of which a different blackening of theX-ray plate is obtained.

X-raying implies certain radiation hazards, but during angiography thecomplication risk is associated in particular with the use of contrastagents.

In recent years, a number of new methods have been introduced indiagnostic radiology. One such method goes by the name NMR (NuclearMagnetic Resonance) which provides information not only about thedistribution of the water content in a specific tissue (incontradistinction to radiology which merely gives a measure of thetransmissibility of the X-rays in a specific tissue), but also about thechemical tissue structure which is different in normal and pathologicaltissue.

In the NMR method, a strong and homogeneous magnetic field is appliedacross the tissue to be examined. By studying the so-called relaxationtimes of the protons of the molecules present, especially the protons ofthe water, it is possible to produce, via comprehensive and complexcomputer calculations, a visual image of the structure of the tissueconcerned.

There is, however, an interest in being able to make a differentialdiagnosis between pieces of tissue having a high density of bloodvessels and, alternatively, tissue having a low density of vessels. Sucha situation which has considerable clinical interest, comprises thelocalisation of tumours which, in their periphery, have a higher densityof vessels as compared with normal tissue.

One useful method in the context is to inject into the vascular systemsome type of particles responsive to a magnetic field and showingchanges in the above-mentioned relaxation times.

These magnetically responsive particles interfere with theabove-mentioned homogeneous magnetic field in that there is formed,around each individual particle, a field gradient which in its turnchanges the relaxation times.

Put more simply, this means that “black holes” are formed around eachparticle which may be visualised and thus give an impression of thevessel density in the tissue in question.

In another diagnostic method, use may be made of the movability ofmagnetically responsive particles in a tissue. The basic principle ofthis method may be studied according to the following: If magneticallyresponsive particle are introduced into a magnetic field, the particleswill align themselves in the direction of the field lines. If the fieldis removed or shut down, the magnetically responsive particles willchange their position in response to processes in the tissue. Theduration of this change may, however, vary between different tissues andalso in dependence upon the localisation of the particles within thetissue in question. This variability of the response of the magneticmaterial may be utilised diagnostically. If magnetically responsiveparticles are administered to a patient, the distribution of theparticles in different organs can be determined by means of a sensitivemagnetometer capable of detecting the above-mentioned changes (Nature(1983) 302, 336).

Ultrasonics is another visualisation technique in which sound-waves arereflected differently against different types of tissue, depending uponthe acoustic impedance of these tissues. Also in this respect, there isan interest in being able to use some type of contrast agent in order toobtain an amplification of specific organs. Particles of different typeshave here been shown to provide changed echo effects and a changedresolution associated therewith (see, Edwards and Jarzynski, J. Acoust.Soc. Am., 74:1006 (1983)). Edwards and Jarzynski studied ultrasoundscattering by spherical microspheres which may contain gas, namely glassbubbles and hydrogen gas bubbles, and discussed future studies withliquid droplets. The present invention includes a sphere or particle foruse in diagnostics or as a contrast agents which agent reflects soundwaves and is characterized in that it consists of a matrix and thediagnostic and/or contrast agent enclosed within said matrix.

It is also possible to use magnetically responsive particles having aCurie point of about 42° C. for use at hyperthermia. In this instance,the magnetically responsive particles are retained during the treatmentof the hyperthermia by a magnetic field, but the moment the tissuetemperature exceeds the Curie point, the particles disappear from thetissue because the magnetic responsiveness disappears at thistemperature.

By labelling the particles with some gamma-radiating nuclide (forexample technetium-99m) it is possible to localise the particles bymeans of a gamma camera and thereby also to combine the examination withsome of the other techniques referred to above.

When using particles within any of the above-mentioned ranges, it isdesired, in order to achieve optimal conditions, to be able to vary theamount of magnetically or otherwise responsive material, withoutaffecting on that account the pharmacodynamic and circulatorycharacteristics of the particles. To be able to do this, one must use atechnique which implies enclosing the responsive material in a matrix,preferably a matrix of spherical shape, and the matrix should per sesatisfy the following criteria:

biocompatible

biologically degradable

nonimmunogenic.

A matrix of this type normally is built up of some type of polymerswhich are held together by chemical bonds. Different types of polymersare available for making such matrices. However, the selection ofpolymers will be very limited if the above-mentioned criteria of thematrix are to be fulfilled.

One type of polymers that has proved useful in these contexts are thecarbohydrates, especially those who are included in the body as anatural constituent.

Endogenous carbohydrates are represented by starch and glycogen, butalso dextran may be included in this group because of its prolonged useas a blood substituent in medical service.

The production of a carbohydrate matrix satisfying these criteria isdescribed in PCT/SE82/00381, which corresponds to U.S. Pat. No.4,501,276, PCT/SE83/00106 which corresponds to U.S. Pat. No. 4,687,748,and PCT/SE83/00268 which corresponds to U.S. Pat. No. 4,713,249.

Another type of polymers that have proved to satisfy the said criteriaare polyamino acids, such as proteins of the type albumin. Theproduction of polyamino acid matrices is disclosed in U.S. Pat. No.4,247,406.

Further types of polymers are represented by synthetic polymers, such asacrylates, polystyrene etc. The production of matrices from syntheticpolymers is well documented in literature.

It is in this connection extremely advantageous if covalentcross-linking of the polymers can be avoided in the production of auseful matrix. For example, covalently cross-linked carbohydratematrices have been found to produce transformed cells, in the form ofgranuloma, when used on humans (Am.Surg. (1955) 142, 1045).

When using covalently cross-linked proteins, there is a risk ofimmunological reactions because the resulting derivatised protein is notrecognised by the body's immunity system as a protein belonging to thebody. There are, however, for specific systems no alternatives to thecovalent cross-linking, especially when using synthetic polymers orcombinations of different polymers and cross-linking agents in order toobtain a useful system. As an example, it is possible to cross-linkacrylic polymers with starch and, alternatively, to cross-link starchwith acrylates.

Another useful possibility which is described in literature is theproduction of larger particles from smaller particles. For example, itis possible to produce, from 0.5 μm particles, conglomerates of largerparticles, for example in the range of about 10 μm.

Another factor of importance to the injection of spheres into thevascular system is that the spheres have a size that prevents them fromgetting stuck in the capillary system of different tissues during thefirst passage. To prevent this, the particle must have a diameter below1 μm (Chem.Pharm.Bull. (1975) 23, 1452) and a surface structure ofhydrophilic character.

When particles are injected into the vascular system, all particles willhave collected after a given period of time in the liver or spleen,(theRES system) because the normal function of these organs is to purify theblood of foreign particles. At present, there is only one methoddescribed which is capable of collecting particles to organs other thanthose mentioned above, and this is by utilising magnetically responsiveparticles or spheres.

This is of particular interest in the context of this invention becausespheres containing magnetically responsive substances can be used and bemade to stop in different tissues by means of the outer magnetic field.When the magnetically responsive particles then are stuck in the desiredtissue, the tissue in question can simply be visualized by means of theNMR method or some of the other techniques referred to above.

DESCRIPTION OF THE INVENTION

The invention relates to responsive particles, preferably spheres, andtheir use as a diagnostic and contrast agent. The invention shows thatit is possible to utilise spheres as contrast agents, the responsivematerial being enclosed within a matrix. The responsive material mayconsist of, for example, magnetite particles enclosed in the form ofdiscrete particles of varying size, or in the form of complexed ions.

One conceivable matrix for use in the context of this invention consistsof carbohydrates that have been stabilised by crystallization, whichmeans that the type of chemical bonds holding the polymeric networktogether is not covalent in character, mainly hydrogen bonds, van derWaals forces or, in some cases, ion bonds.

As carbohydrate, use may be made of all conceivable variants, includingcarbohydrates of varying molecular weight and/or substituted orotherwise derivatised carbohydrates. For example, it may be mentionedthat it is possible to produce and use magnetically responsivecarbohydrate particles in which the carbohydrate is of starch origin andlow-molecular of the type glucose, maltose, dextrins etc., andsuccessively increasing molecular weight up to native potato starchhaving a molecular weight of several millions. The same molecular weightrange is applicable to other carbohydrate groups, such as dextran orglycogen.

Another matrix for use in the complex of this invention may consist ofpolyamino acids, such as the protein albumin in which the matrix isstabilised by heating and the cohering forces are not covalent incharacter, of the type hydrophobic interactions, hydrogen bonds, van derWaals forces or ion bonds. In a manner similar to what has been statedabove, it is also possible to use synthetic polymers or combinations asmatrix.

The following Example should not be regarded as restrictive and merelyserves to illustrate the main features of the invention.

EXAMPLE

Dextran spheres having a size of about 1 μm with enclosed magnetiteparticles (size 10-20 nm) were suspended in human serum. The relaxationtimes of the solution were measured with an NMR apparatus (Praxis II,Alnor Instrument AB, Nyköping) and compared with the relaxation timesfor the same serum without magnetically responsive dextran spheres. Thefollowing values of T1 and T2, respectively, were obtained.

T1 (ms) T2 (ms) Serum without particles: 1660 400 Serum with particles:conc.: 0.05 mg/ml 1342 109 0.1 mg/ml 1306 82.2 0.2 mg/ml 1147 52.6 0.5mg/ml 968 30.7 1.0 mg/mi 813 24.0 2.0 mg/ml 688 19.9 4.0 mg/ml 691 22.9

What is claimed is:
 1. A method of contrast enhanced ultrasonicdiagnostic imaging comprising: administering to a subject a contrastenhancing amount of spherical particles comprising a matrix of abiocompatible, biodegradable, non-immunogenic polymer enclosing agaseous contrast agent which reflects sound waves, said polymer beingselected from the group consisting of carbohydrates, carbohydratederivatives and non-polyamino acid synthetic polymers; and generating anultrasonic image of said subject.
 2. The method of claim 1 wherein thematrix is of spherical shape.
 3. The method of claim 1 wherein thematrix is stabilized by non-covalent bonds.
 4. The method of claim 1wherein the matrix comprises a polymer held together by chemical bonds.5. The method of claim 1 wherein the matrix comprises a carbohydrate orcarbohydrate derivative.
 6. The method of claim 5 wherein thecarbohydrate is endogenous.
 7. The method of claim 5 wherein the matrixcomprises glucose, maltose, sucrose, sorbitol or dextrin.
 8. The methodof claim 5 wherein the carbohydrate or carbohydrate derivative is amonosaccharide or a polysaccharide having two or more saccharide units.9. The method of claim 8 wherein the carbohydrate comprises starch,glycogen or dextran.
 10. The method of claim 4 wherein the polymercomprises a synthetic polymer.
 11. The method of claim 10 wherein thesynthetic polymer is selected from the group consisting of acrylates,polystyrene and derivatives thereof.
 12. The method of claim 10 whereinthe synthetic polymer is covalently crosslinked.
 13. The method of claim10 wherein the synthetic polymer comprises an acrylic polymercrosslinked with starch.
 14. The method of claim 10 wherein thesynthetic polymer comprises starch crosslinked with acrylates.
 15. Themethod of claim 1 wherein the spherical or particles have a surfacestructure of hydrophilic character.
 16. The method of claim 1 whereinthe spherical or particles have a diameter in the range of 0.01-1000 μm.17. The method of claim 1 wherein the spherical or particles have adiameter less than 1 μm.
 18. The method of claim 1 wherein the particlesare built up of conglomerates of smaller particles or spherical.
 19. Themethod of claim 18 wherein the size of the conglomerates is about 10 μm.20. The method of claim 18 wherein the size of the smaller particles orspheres is about 0.5 μm.
 21. The method of claim 1 wherein an ultrasonicimage is generated of the reticuloendothelial system, including theliver and spleen, of said subject.
 22. The method of claim 1 comprisingthe further step of collecting particles to organs by additionallyutilizing magnetically responsive particles or spheres and an outermagnetic field.